Stanislav Kopriva

Sulfur Assimilation in Photosynthetic Organisms: Molecular Functions and Regulations of Transporters and Assimilatory Enzymes

Sulfur is required for growth of all organisms and is present in a wide variety of metabolites having distinctive biological functions. Sulfur is cycled in ecosystems in nature where conversion of sulfate to organic sulfur compounds is primarily dependent on sulfate uptake and reduction pathways in photosynthetic organisms and microorganisms. In vascular plant species, transport proteins and enzymes in this pathway are functionally diversified to have distinct biochemical properties in specific cellular and subcellular compartments. Recent findings indicate regulatory processes of sulfate transport and metabolism are tightly connected through several modes of transcriptional and posttranscriptional mechanisms. This review provides up-to-date knowledge in functions and regulations of sulfur assimilation in plants and algae, focusing on sulfate transport systems and metabolic pathways for sulfate reduction and synthesis of downstream metabolites with diverse biological functions.

The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants

The availability of nitrogen varies greatly in the ocean and limits primary productivity over large areas. Diatoms, a group of phytoplankton that are responsible for about 20% of global carbon fixation, respond rapidly to influxes of nitrate and are highly successful in upwelling regions. Although recent diatom genome projects have highlighted clues to the success of this group, very little is known about their adaptive response to changing environmental conditions. Here, we compare the proteome of the marine diatom Thalassiosira pseudonana (CCMP 1335) at the onset of nitrogen starvation with that of nitrogen-replete cells using two-dimensional gel electrophoresis. In total, 3,310 protein spots were distinguishable, and we identified 42 proteins increasing and 23 decreasing in abundance (greater than 1.5-fold change; P < 0.005). Proteins involved in the metabolism of nitrogen, amino acids, proteins, and carbohydrates, photosynthesis, and chlorophyll biosynthesis were represented. Comparison of our proteomics data with the transcriptome response of this species under similar growth conditions showed good correlation and provided insight into different levels of response. The T. pseudonana response to nitrogen starvation was also compared with that of the higher plant Arabidopsis (Arabidopsis thaliana), the green alga Chlamydomonas reinhardtii, and the cyanobacterium Prochlorococcus marinus. We have found that the response of diatom carbon metabolism to nitrogen starvation is different from that of other photosynthetic eukaryotes and bears closer resemblance to the response of cyanobacteria.

Disruption of adenosine-5'-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites

Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 5′-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate assimilation pathway or phosphorylated by APS kinase to 3′-phosphoadenosine 5′-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normal growth rates.

Interplay of SLIM1 and miR395 in the regulation of sulfate assimilation in Arabidopsis

MicroRNAs play a key role in the control of plant development and response to adverse environmental conditions. For example, microRNA395 (miR395), which targets three out of four isoforms of ATP sulfurylase, the first enzyme of sulfate assimilation, as well as a low-affinity sulfate transporter, SULTR2;1, is strongly induced by sulfate deficiency. However, other components of sulfate assimilation are induced by sulfate starvation, so that the role of miR395 is counterintuitive. Here, we describe the regulation of miR395 and its targets by sulfate starvation. We show that miR395 is important for the increased translocation of sulfate to the shoots during sulfate starvation. MiR395 together with the SULFUR LIMITATION 1 transcription factor maintain optimal levels of ATP sulfurylase transcripts to enable increased flux through the sulfate assimilation pathway in sulfate-deficient plants. Reduced expression of ATP sulfurylase (ATPS) alone affects both sulfate translocation and flux, but SULTR2;1 is important for the full rate of sulfate translocation to the shoots. Thus, miR395 is an integral part of the regulatory circuit controlling plant sulfate assimilation with a complex mechanism of action.

Natural variation for sulfate content in Arabidopsis thaliana is highly controlled by APR2.

Most agronomic traits of importance, whether physiological (such as nutrient use efficiency) or developmental (such as flowering time), are controlled simultaneously by multiple genes and their interactions with the environment. Here, we show that variation in sulfate content between wild Arabidopsis thaliana accessions Bay-0 and Shahdara is controlled by a major quantitative trait locus that results in a strong interaction with nitrogen availability in the soil. Combining genetic and biochemical results and using a candidate gene approach, we have cloned the underlying gene, showing how a single–amino acid substitution in a key enzyme of the assimilatory sulfate reduction pathway, adenosine 5′-phosphosulfate reductase, is responsible for a decrease in enzyme activity, leading to sulfate accumulation in the plant. This work illustrates the potential of natural variation as a source of new alleles of known genes, which can aid in the study of gene function and metabolic pathway regulation. Our new insights on sulfate assimilation may have an impact on sulfur fertilizer use and stress defense improvement.

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide andO-acetyl-l-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. UsingLemna minor, we analyzed the effects of omission of CO2 from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5′-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO2 led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO2 on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO2, APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO2 also recovered both enzyme activities, with OAS again influenced only APR.35SO4 2− feeding showed that treatment in air without CO2 severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of 35S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of 35S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.

Adenosine 5′-Phosphosulfate Sulfotransferase and Adenosine 5′-Phosphosulfate Reductase Are Identical Enzymes

Adenosine 5′-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M r of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K m = 6.5 μm) and not adenosine 3′-phosphate 5′-phosphosulfate as sulfonyl donor. TheV max of recombinant Lemna APS sulfotransferase (40 μmol min−1 mg protein−1) was about 10 times higher than the previously published V max of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO3 2−. Bound sulfite in the form ofS-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO3 2− was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.

Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis thaliana

The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467 selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time course from 30 min to 24 h. 97 and 64 genes were identified that were up- or down-regulated more than 2–fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfur-related genes were by far strongest affected. Gene expression and metabolite patterns of sulfur metabolism were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response. These results document for the first time how comprehensively the regulation of sulfur-related genes and plant defense are connected. This interaction is discussed as a new approach to differentiate between supply- and demand-driven regulation of the sulfate assimilation pathway.

Arabidopsis root growth dependence on glutathione is linked to auxin transport.

Glutathione depletion, e.g. by the inhibitor of its synthesis, buthionine sulphoximine (BSO), is well known to specifically reduce primary root growth. To obtain an insight into the mechanism of this inhibition, we explored the effects of BSO on Arabidopsis root growth in more detail. BSO inhibits root growth and reduces glutathione (GSH) concentration in a concentration-dependent manner leading to a linear correlation of root growth and GSH content. Microarray analysis revealed that the effect of BSO on gene expression is similar to the effects of misregulation of auxin homeostasis. In addition, auxin-resistant mutants axr1 and axr3 are less sensitive to BSO than the wild-type plants. Indeed, exposure of Arabidopsis to BSO leads to disappearance of the auxin maximum in root tips and the expression of QC cell marker. BSO treatment results in loss of the auxin carriers, PIN1, PIN2 and PIN7, from the root tips of primary roots, but not adventitious roots. Since BSO did not abolish transcription of PIN1, and since the effect of BSO was complemented by dithiothreitol, we conclude that as yet an uncharacterised post-transcriptional redox mechanism regulates the expression of PIN proteins, and thus auxin transport, in the root tips.

Identification of a Pentatricopeptide Repeat Protein Implicated in Splicing of Intron 1 of Mitochondrial nad7 Transcripts

Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis.